Traditional plant breeding strategies to develop new lines of plants that exhibit particular traits are time consuming and sometimes unpredictable. Existing strategies, such as Agrobacterium-mediated transformation and particle bombardment depend heavily on the tissue and genotype. Cell penetrating peptides (CPPs) are a novel and fast growing class of short peptides that are known to play an important role in translocation of a wide range of cargo complexes including DNA, RNA and proteins across the cell membranes in mammalian and human cell lines (Schwartz and Zhang, 2000; Langel, 2002; Vives, 2002).
While CPPs have been shown to facilitate cargo delivery in mammalian cells, the use of CPP in plant cells for transfection studies has been limited by a number of factors. A major obstacle to adapting this technology to plants is that, unlike animal cells, plant cells present a dual barrier system (cell wall and plasma membrane) for the internalization of CPPs and their cargos. Therefore, CPPs must overcome these two barriers for efficient translocation of cargo molecules into intact plant cells. CPPs have been used in plant cells but have relied on the use of permeabilization agents to effectuate the delivery of cargo molecules into intact plant cells. CPP-mediated delivery of small molecules, nucleic acids and proteins into intact plant cells remains largely unexplored and is advantageous for in vitro and in vivo genetic and biochemical manipulations in plant systems.
Nanoparticles have unique properties that have been exploited for use in the delivery of DNA to cells. Metal nanoparticles, such as gold (Au) nanoparticles have been used for DNA delivery because of their low cytotoxicity and ease of functionalization with various ligands of biological significance. In addition to metal nanoparticles, semi-conductor nanoparticles (e.g., quantum dots) (“QD”) within the size range of 3-5 nm have also been used as carriers to deliver molecules into cells. DNA and proteins can be linked to the ligand attached to the QD via various surface functionalizations (see, e.g. Patolsky, F. et al., J. Am. Chem. Soc. 125, 13918 (2003)).
Nanoparticles have been used to deliver plasmid DNA to a variety of animal cells. It has been found that when DNA coated nanoparticles are incubated with cells not having a cell wall, the cells take up the nanoparticles and begin expressing any genes encoded on the DNA. However, the contemporary plant gene delivery is challenging due to the presence of plant cell walls, which leads to the common reliance on invasive delivery means for genetic transformation of plants. Where nanoparticles delivery to cells normally having a cell wall is desired, the cell's wall is stripped before the addition of the particles to protoplasts of plant (see, Torney, F. et al., Nature Nanotechnol. 2, (2007)). In plant cells, the cell wall presents a formidable barrier for the delivery of exogenously applied molecules. Many invasive methods, like gene gun (biolistics), microinjection, electroporation, and Agrobacterium, have been employed to achieve gene and small molecule delivery into walled plant cells, but delivery of proteins has only been achieved by microinjection.
With the ever-growing information from the plant genome-sequencing projects there is an urgent need for a fast, universal (tissue/genotype independent) method in plants for functional genomic studies of a wide array of genes and for the development of transgenic plants expressing important agronomic traits.